Methods for treating parkinson&#39;s disease

ABSTRACT

The present invention relates to a method for treating Parkinson&#39;s disease. According to this method, a halorhodopsin protein, a polynucleotide encoding the halorhodopsin protein or a vector containing the polynucleotide above is treated to ventrolateral thalamus (VL) or medial globus pallidus (GPm) neurons of a subject having Parkinson&#39;s disease, followed by illumination with green light or a T type Ca 2+  channel blocker is treated to VL neurons of a subject having Parkinson&#39;s disease to inhibit rebound firing of VL neurons. By inhibiting rebound firing of VL neurons, Parkinson&#39;s disease can be treated or prevented.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/962,894, filed Apr. 25, 2018, which is herein incorporated byreference in its entirety.

INCORPORATION OF ELECTRONIC SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file, created on Oct.21, 2020, 97.2 KB, which is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a method for treating Parkinson'sdisease comprising the step of inhibiting rebound firing ofventrolateral thalamus (VL) neurons.

2. Description of the Related Art

Parkinson's disease is a progressive disease taking the second highestincidence among neurodegenerative diseases. The incidence rate of thisdisease is continuously increasing with the increase of the agedpopulation, so that it is a socially and economically problematicintractable disease. It is known that approximately 4 million peopleworldwide are suffering this disease. In USA, about 50,000 new patientshave been reported every year. The incidence rate of this disease is onein 1000 people, and the higher the age, the higher the incidence.

The exact cause of Parkinson's disease has not been disclosed yet, butit is believed that the disorder in the neurons gathered tightly in parscompacta of substantia nigra of basal ganglia is the reason. Theseneurons produce dopamine, a neurotransmitter. Dopamine is functioning asan inhibitor of nerve stimulation in the brain so that it is involved insuppressing unintentional movement. It is also involved in regulatingsignal output of globus pallidus through caudate nucleus and putamen. Inthe case of Parkinson's disease, as dopamine neurons in the substantianigra die, their signaling is reduced and inhibitory signaling throughthe D1 receptor of the striatum is also reduced. In general, when theglobus pallidus suppresses the thalamus excessively, the motor neuronsthat descend from the thalamus to the cerebral cortex are suppressed tocause Parkinson's disease specific symptoms such as bradykinesia.

The drugs currently used or under development for the treatment ofParkinson's disease are as follows. The most widely used drugs aredopamine precursors and dopamine receptor fenofibrates such as Levodopa.In addition, COMT inhibitors and MAO-B inhibitors functioning tomaintain the level of dopamine in the brain by suppressing dopaminemetabolism have been used. Antimuscarinics and NMDA antagonists havebeen developed and used as drugs for improving neurotransmitters otherthan dopamine. The attempts to develop or use brain cell protectiveagents, antioxidants, brain cell death inhibitors, and brain functionagonists as therapeutic agents have been made. To treat the terminalpatients who cannot be efficiently treated by pharmacotherapy, asurgical operation such as deep brain stimulation is tried. However,since the cause of Parkinson's disease is not known exactly, thetreatment methods of these days are only to improve the symptoms insteadof fundamentally treating the disease.

Optogenetics is a technology that combines optics and genetics, which isa biological technique to regulate cells of living tissues with light.The most representative example in this field is that neurons aregenetically manipulated in order to express ion channels that respond tolight. By using optogenetics, the activity of each individual neuron inliving tissues or even in free-moving animals can be regulated andobserved and also the effect of the regulation of neuronal activity canbe observed in real time. To regulate the neuronal activity,light-responsive proteins such as channelrhodopsin, halorhodopsin, andarchaerhodopsin can be used. To record the neuronal activity optically,optogenetic sensors such as GCaMP sensing the changes of calciumconcentration, synaptopHluorin sensing the secretion of neurons, andArclightning (ASAP1) sensing the cell membrane potential are used. Asthe regulation of neuronal activity is realized by taking advantage ofoptogenetics, it can be applied to understand the mechanism ofneurological disease or to develop a new treatment method forneurological disease.

The intracellular calcium influx through voltage-gated calcium channelis known to mediate a wide range of cellular and physiological responsesincluding hormone secretion and gene expression. The voltage-gatedcalcium channel is deeply involved in the secretion and transmission ofneurotransmitters and is mainly found in the central and peripheralnervous system and neuroendocrine cells. The voltage-gated calciumchannel is classified into L type, T type, N type, P/Q type, and R typein mammalian cells. The T-type calcium channel has three subtypes calledα1G(Ca_(v)3.1), α1H(Ca_(v)3.2), and α1I(Ca_(v)3.3). According to theprevious reports, T-type calcium channels are involved in pathologiesrelated to neurological diseases and disorders including epilepsy,essential hypertension, pain, neuropathic pain, schizophrenia,Parkinson's disease, depression, anxiety, sleep disorder, dyspnoea,psychosis, and schizophrenia (references: J Neuroscience, 14, 5485(1994); Drugs Future 30(6), 573-580(2005); EMBO J, 24, 315-324 (2005);Drug Discovery Today, 11, 5/6, 245-253(2006)).

The attempt to treat Parkinson's disease using optogenetics or T-typecalcium channel was tried but this was only based on the technique toalleviate the inhibitory signals of the basal ganglia that suppressmotor neurons. The present inventors confirmed that the inhibitory inputfrom the medial globus pallidus (GPm) produced excitatory motor signalsexcessively after the suppression via T-type calcium channels in theventrolateral thalamus (VL) and such excitatory motor signalsaccelerated abnormal movements similar to Parkinson's disease.Thereafter, the present inventors confirmed that when either inhibitoryinputs from the GPm or postsynaptic VL neurons were photoinhibited orthe T-type calcium channel involved in inducing rebound firing in theventrolateral thalamus was blocked or knocked-down, the excitatory motorsignal of the thalamus was inhibited and the movement abnormalitiessimilar to Parkinson's disease was alleviated in Parkinson's diseaseanimal model, leading to the completion of the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fortreating Parkinson's disease.

To achieve the above object, the present invention provides a method fortreating Parkinson's disease comprising the step of inhibiting reboundfiring of ventrolateral thalamus (VL) neurons.

Advantageous Effect

In the method for treating Parkinson's disease of the present invention,a halorhodopsin protein, a polynucleotide encoding the halorhodopsinprotein or a vector containing the polynucleotide above is treated toventrolateral thalamus (VL) or medial globus pallidus (GPm) neurons of asubject having Parkinson's disease, followed by illumination with greenlight, or a Ca_(v)3.1 gene expression inhibitor is treated to VL neuronsof a subject having Parkinson's disease to inhibit rebound firing of VLneurons. By inhibiting rebound firing of VL neurons, Parkinson's diseasecan be treated or prevented.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1(A) illustrates the experimental scheme for photostimulation ofGPm-VL synapses and recording activities from VL neurons and bodymuscles, and FIG. 1(B illustrates the expression of the cation channel,ChR2, in GPm neurons (white arrows and GPm axon terminals in the VLthalamus (Scale bars: 60 μm (GPm) and 100 μm (VL)).

FIG. 2(A) illustrates the movement of the mouse before and after (bluerectangle) photostimulation on the GPm-VL synapse, and FIG. 2(B)illustrates the quantification of the photostimulation effect onlocomotion (Blue bars: 15 s photostimulation (20 Hz, 5 ms pulse width)).

FIG. 3 illustrates the representative traces of photo-induced EMGsignals in WT mice (Blue bars: Light stimulation (1 Hz, 50 ms pulsewidth; 5 Hz, 50 ms pulse width; 20 Hz, 5 ms pulse width); Green arrows:Slow muscle twitching; Red arrows: Rapid muscle twitching).

FIG. 4 illustrates the representative photo-induced EMG signal from a WTmouse (left) and band-pass filtered EMG activity in each frequency range(right)(Rectified EMG signals (filled line plot) showingphotostimulation-induced muscle activity. The frequency distribution ofEMG activity is presented as a pseudocolor spectrogram. Three colorasterisks in the spectrogram indicate the frequency ranges with clearincreases in EMG amplitude).

FIG. 5 illustrates the time-series images of WT mice during thehorizontal bar test (left) and the average latency to release with nophotostimulation (OFF) and 20 Hz photostimulation (right).

FIG. 6(A) illustrates the multi-unit activity of VL neurons (VL), thelocal field potentials in primary motor cortex (M1-LFP), and the EMGrecordings from muscles (neck and arm EMG) after photostimulation (5 Hz,50 ms pulse width) in WT mice; and FIG. 6(B) illustrates the same datain CaV3.1-KO mice (Asterisks: absence of muscle responses; Red arrows:activated MUA signals; Blue bars: time for light-On).

FIG. 7 illustrates the comparison of the average firing of multi-unitactivity (MUA) or VL neurons before and during 473 nm light (1 Hz, 50-mspulse width) between WT and Ca_(v)3.1-KO mice.

FIG. 8(A), FIG. 8(B), FIG. 8(D) and FIG. 8(E) illustrate the rasterplots of multi-unit activity (MUA) and single-unit activity (SUA) of VLneurons before and after photostimulation with 473 nm light (1 Hz, 50-mspulse width) in WT (FIG. 8(A) and FIG. 8(D)) and CaV3.1-KO (FIG. 8(B)and FIG. 8(E)) neurons (Each horizontal row shows an individual trial inthe VL area.).

FIG. 8(C) illustrates the earlier onset and higher rebound firing ratein WT than KO neurons.

FIG. 8(F) illustrates the no significant differences in peak firingrates between genotypes (Red arrows: time of peak firing; Gray dottedline: baseline firing rate).

FIG. 9 illustrates the fold changes in peak firing rate after inhibitionmeasured using SUA and MUA.

FIG. 10 illustrates the comparison of cumulative response probability (%total neurons recorded) based on latency to peak rebound firing rate forWT and KO mice (LP50 is the time at which 50% neurons exhibit peakrebound firing).

FIG. 11(A) illustrates the IPSPs evoked by 488 nm photostimulation ofGPm inputs recorded in patch-clamped VL neurons, and FIG. 11(B) and FIG.11(C) illustrate the similar probability of IPSP response FIG. 11(B) andthe similar mean IPSP amplitudes FIG. 11(C) in WT and CaV3.1-KO micefollowing photostimulation.

FIG. 12(A) and FIG. 12(B) illustrate the rebound firing in VL neurons ofWT mice before and after Ni2+ treatment, and FIG. 12(C) and FIG. 12(D)illustrate the raster plots of action potentials after hyperpolarizationby injection in thalamic slices from WT mice before and after Ni2+treatment (Red arrows: First spike after hyperpolarization).

FIG. 13(A) and FIG. 13(B) illustrate the latency to first spike, andFIG. 13(C) and FIG. 13(D) illustrate the onset variation of the firstspike before and after Ni2+ treatment and between WT and CaV3.1-KOneurons in brain slices.

FIG. 14 illustrates the distribution of neurons classified by latency tofirst-spike onset after photostimulation (Black and red dotted linesshow cumulative response probability by latency to first-spike onset).

FIG. 15(A) illustrates the representative rebound slope, and FIG. 15(B)illustrates the differences in membrane potential before and after Ni2+treatment in brain slices (WT: black; KO: red).

FIG. 16(A) illustrates the induction of rebound firing dependent on thepulse width (5, 25 or 50 ms) of photostimulation in WT; and FIG. 16(B)illustrates the same results in CaV3.1-KO (Arrows: induction of reboundfiring (red) and muscle responses (blue); Asterisks: absence of reboundfiring and muscle responses).

FIG. 17(A) illustrates the photostimulation-induced akinesia-like motorabnormalities and tremor-like motor abnormalities in behaving WT; FIG.17(B) illustrates the same data in CaV3.1-KO mice.

FIG. 18 illustrates the experimental scheme for photoactivation ofGPm-VL synapses and photoinhibition of VL somata.

FIG. 19 illustrates the sparse expression of green light-responsiveeNpHR3.0 in VL somata (Green: eNpHR3.0; Blue: DAPI).

FIGS. 20(A), 20(B) and 20(C) illustrate the representative EMG responsesto photoinhibition of VL somata with photoactivation of GPm-VL synapses.FIG. 20(A): Photoactivation of GPm-VL synapses with no photoinhibitionof VL somata (None); FIG. 20(B): Photoinhibition of VL somata within 200ms GPm-VL pathway photoactivation (Early); and FIG. 20(C):Photoinhibition of VL somata during 250-500 ms after GPm-VL pathwayphotoactivation (Late).

FIG. 21 illustrates the comparison of muscle activation inphotoactivation of GPm-VL synapses with no photoinhibition of VL somata(None), photoinhibition of VL somata within 200 ms GPm-VL pathwayphotoactivation (Early) and photoinhibition of VL somata during 250-500ms after GPm-VL pathway photoactivation (Late).

FIG. 22(A) illustrates the quantification of the effect of GPm-VLphotostimulation with blue light on locomotion, and FIG. 22(B)illustrates the quantification of the dual stimulation effect with bluelight (for activating hChR2 in axon terminals from GPm to VL thalamus)and green light (for activating eNpHR3.0 in VL somata) on locomotion.

FIG. 23(A) illustrates the raster plots depicting increased spikingactivity of VL neurons in WT and FIG. 23(B) illustrates the same data inSPR-KO mice following spontaneous inhibitory events (Pink box: the 50 msepoch preceding the spontaneous inhibitory event; Red arrows: reboundfiring after the spontaneous inhibitory event).

FIG. 24 illustrates the quantification of spikes following spontaneousinhibitory events in VL neurons of WT and SPR-KO mice.

FIG. 25 illustrates the experimental scheme for photoinhibition ofGPm-VL synapses while recording the activities of VL neurons in WT andSPR-KO mice and expression of eNpHR3.0 protein in somata of GPm neuronsand their axon terminals in the VL thalamus.

FIG. 26 (A) illustrates the multi-unit and single unit activity and Foldchange in rebound spiking activity of VL neurons in SPR-KO micefollowing spontaneous inhibitory events before and after photoinhibitionof GPm-VL synapses (Green rectangle: 532 nm continuous light for GPmphotoinhibition).

FIG. 26(B) and FIG. 26(C) respectively illustrate the comparison of theVL neuronal activities and Comparison of the rebound firing in MUA andLFP before and after the ethosuximide treatment in SPR-KO mice.

FIG. 27(A) illustrates the percentage of time spent moving duringlight-ON (10 s) and light-OFF (10 s) periods in SPR-KO mice, and FIG.27(B) and FIG. 27(C) illustrate the photoinhibition of GPm-VL synapsesin SPR-KO mice suppresses movement initiation delay FIG. 27(B) andakinesia FIG. 27(C).

FIG. 28 illustrates the photoinhibition of GPm-VL synapses reducesrigidity of SPR-KO mice measured in a horizontal bar test (The greenrectangle: the period of photoinhibition (20 s, continuous 532 nmlight)).

FIG. 29(A) illustrates the reduction of tremor activity duringphotoinhibition, FIG. 29(B) illustrates the comparison of the tremorscore between ethosuximide treated (ETX) and saline treated (Sal)control mice, and FIG. 29(C) illustrates the comparison of the tremorscore between the mice with VL-specific CaV3.1 knockdown (Sh3.1) and thecontrol mice with scramble virus (Scr).

FIG. 30 illustrates the schematic depiction of the experimental setupfor recording VL thalamic activity during natural resting-running(Nogo-Go or Go-Nogo) transitions.

FIG. 31 illustrates the firing activities of VL neurons arranged indescending order of the selectivity index expressing the association ofspiking with movement(selectivity index=(fmovementfresting)/(fmovement+fresting)) (Each horizontal row presents theactivity of individual VL neurons 3 s before and after the transition.The neurons in upper rows showed higher movement-related neuralactivities).

FIG. 32(A), FIG. 32(B), FIG. 32(C) and FIG. 32(D) illustrate thealignment of multi-unit spikes for 50 ms preceding and following aninhibitory event defined as a reduction in baseline firing frequency of≥50% during one of the four possible behavioral states (FIG. 32(A) Nogo,FIG. 32(B) Nogo-Go, FIG. 32(C) Go, FIG. 32(D) Go-Nogo).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail.

The present invention provides a method for treating Parkinson's diseasecomprising the step of inhibiting rebound firing of ventrolateralthalamus (VL) neurons.

The rebound firing of VL neurons above is induced by the inhibitoryinput transmitted from the medial globus pallidus (GPm), and the reboundfiring can be inhibited by photoinhibition of VL or GPm neurons.

To inhibit the rebound firing of VL neurons, a polynucleotide encoding ahalorhodopsin protein or a vector containing the polynucleotide abovecan be introduced in ventrolateral thalamus (VL) or medial globuspallidus (GPm) neurons of a subject having Parkinson's disease.

The said halorhodopsin protein responses to light to flow chloride intocells to cause hyperpolarization, indicating it plays a role as an ionpump. The halorhodopsin protein is composed of the amino acid sequencerepresented by SEQ. ID. NO: 1.

The said halorhodopsin protein preferably responses to light to flowchloride ions (Cl⁻) into cells to cause hyperpolarization therein andaccordingly inhibits the rebound firing of VL neurons. Any protein thatcan inhibit the rebound firing of VL neurons by responding to light canbe used herein without limit. Particularly, the protein can be iC⁺⁺ orArchT, but not always limited thereto.

The said halorhodopsin protein can include not only a wild type proteinhaving an activity of a chloride ion pump but also a functionalhomologue displaying an activity of a chloride ion pump with at least90% amino acid homology.

In this invention, the term “homology” indicates the level of similarityto the amino acid sequence of a wild type protein. The halorhodopsinprotein of the present invention comprises an amino acid sequence havingat least 70% homology, preferably at least 90% homology, and morepreferably at least 95% homology with the wild type amino acid sequencerepresented by SEQ. ID. NO: 1. The comparison of homology can beperformed by observing with the naked eye or by using a comparisonprogram that is easy to purchase. The commercially available computerprogram can calculate homology between two or more sequences as % andhomology (%) can be calculated for adjacent sequences.

The said halorhodopsin protein can include an amino acid sequencevariant thereof as long as it retains the activity of a chloride ionpump. The variant herein indicates the protein having a differentsequence from the natural amino acid sequence due to deletion,insertion, non-conservative or conservative substitution of one or moreamino acid residues, or a combination thereof.

Such a variant includes a functional homologue having an equivalentactivity to the wild type or a modified protein having modificationsthat increase or decrease physicochemical properties. Preferably, theprotein herein is a variant in which the physicochemical properties aremodified. For example, the variant herein has the increased structuralstability against the external environment including physical factorssuch as temperature, moisture, pH, electrolyte, reducing sugar,pressurization, drying, freezing, interfacial tension, light, repetitionof freezing and thawing and high concentration, and chemical factorssuch as acid, alkali, neutral salt, organic solvent, metal ion,oxidation-reduction agent, and protease. It can also be a variant withthe increased activity due to the modification in the amino acidsequence.

The said halorhodopsin protein can be directly isolated from livingorganisms, chemically synthesized, or obtained by using geneticrecombination techniques. In the case of isolating the halorhodopsinprotein directly from living organisms, the isolation and purificationof the halorhodopsin protein contained in cells can be performed byvarious generally known methods. In the case of synthesizing the proteinchemically, a polypeptide synthesis method well known to those in theart can be used. The Polypeptide can be prepared by using theconventional stepwise liquid or solid phase synthesis, fractionalcondensation, F-MOC or T-BOC chemical method. In the case of usinggenetic recombination techniques, the polynucleotide (nucleic acid)encoding the halorhodopsin protein is inserted in a proper expressionvector, which is introduced in a host cell for transfection, and thenthe host cell is cultured to express the halorhodopsin protein, followedby collecting the protein from the host cell. The protein is expressedin selected host cells and then purified by the conventional biochemicalseparation techniques such as treatment with a protein precipitant(salting-out), centrifugation, ultrasonic disruption, ultrafiltration,dialysis, molecular sieve chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, and affinitychromatography. To separate the protein with high purity, those methodsare used in combination.

The polynucleotide encoding the halorhodopsin protein can be composed ofthe nucleotide sequence represented by SEQ. ID. NO: 2, or can be apolynucleotide in which one or more nucleotide sequences capable ofencoding the active halorhodopsin protein are substituted, deleted, orinserted. The polynucleotide in which one or more nucleotide sequencesare substituted, deleted, or inserted can have at least 70% homology,preferably at least 80% homology, and more preferably at least 90%homology with the polynucleotide represented by SEQ. ID. NO: 2.

The vector containing the polynucleotide above can contain a cloningorigin, a promoter, a marker gene, and a translation regulatory element.The said vector can be a gene construct comprising an essentialregulatory element operably linked thereto in order to express the geneinsert so that a target protein can be expressed in proper host cells.

The vector containing the polynucleotide above can be selected from thegroup consisting of a linear DNA vector, a plasmid DNA vector, and arecombinant viral vector. The recombinant viral vector can be selectedfrom the group consisting of retrovirus, adenovirus, adeno-associatedvirus, and lentivirus. In a preferred embodiment of the presentinvention, the vector containing the polynucleotide is preferablyadeno-associated virus, but not always limited thereto.

The vector containing the polynucleotide can be introduced inventrolateral thalamus (VL) or medial globus pallidus (GPm) neurons viaone of the methods selected from the group consisting of transfection,electroporation, transduction, microinjection, and ballisticintroduction. According to an embodiment of the present invention,transfection is most preferably used but not always limited thereto.

The method of the present invention can additionally include a step ofirradiating green light to a subject introduced with the halorhodopsinprotein, the polynucleotide encoding the halorhodopsin protein, or thevector containing the polynucleotide above.

The green light above has a wavelength of 480 to 550 nm, and preferably500 to 550 nm. According to an embodiment of the present invention, thegreen light can have a wavelength of 532 nm.

The step of irradiating green light is to irradiate postsynaptic VLneurons.

In a preferred embodiment of the present invention, medial globuspallidus (GPm) neurons were infected with the adeno-associated virus(AAV) vector containing channelrhodopsin-2 (hChR2) gene andphotostimulated to investigate the effect of inhibitory input of thebasal ganglia (BG) on the thalamus. As a result, it was confirmed thatthe GPm-VL circuit plays an important role in the suppression of mouselocomotion (see FIGS. 1 and 2).

To examine whether the GPm-VL circuit affects the generation of motorsignals, electromyography was performed. As a result, it was confirmedthat the medial globus pallidus (GPm) inhibitory input to the VL aloneis sufficient to trigger signals for muscle contraction, potentiallyleading to various motor responses, including suppressed locomotoractivity, tremor, and rigidity, and the magnitude of this activityvaried with the frequency of photostimulation (see FIGS. 3-5).

The present inventors also observed the activity of neurons in VL andmotor cortex (M1), as well as the muscle activity duringphotostimulation of the GPm-VL input. As a result, it was confirmed thatthe activation of GPm-VL inhibitory synapses induces rebound firing ofVL neurons, which stimulates the motor cortex (M1) (see FIG. 6).

The present inventors also investigated the time course of actionpotential firing in VL neurons in response to photostimulation of GPm-VLinputs. As a result, VL neurons evoked a surge of rebound firing within200 ms of the post-inhibitory period and the early-onset surge ofrebound firing seems to depend on the number VL neurons (see FIGS.8˜10).

The present inventors designed an optogenetic experiment and performedin which halorhodopsin (eNpHR3.0) was expressed in the VL to facilitatephotoinhibition of VL neurons while channelrhodopsin-2 (hChR2) wasexpressed in the medial globus pallidus (GPm) to allow photostimulationof GPm inhibitory inputs to the VL. As a result, it was confirmed thatthe photoactivation of GPm-VL inputs robustly induced muscularcontractions, and this motor response was abolished by postsynapticphotoinhibition during the early (<200 ms) post-inhibitory period (seeFIGS. 18-21). It was also confirmed that the number of neurons thatevoke rebound firing during the early post-inhibitory period controlsthe amount of excitatory output from the VL.

The present inventors also confirmed that hypokinesia caused byphotostimulation of GPm-VL inputs was efficiently restored byphotoinhibition of VL neurons after the photostimulation of GPm-VLinputs (see FIG. 22).

The present inventors constructed a mouse model of dopamine deficiency(SPR-KO) exhibit Parkinson's disease like motor abnormalities, followedby investigation of spontaneous rebound firing. As a result, it wasconfirmed that the dopamine-deficiency induced inhibition appears toevoke rebound firing in a PD-like mouse model (see FIGS. 23 and 24).

The present inventors induced the expression of halorhodopsin in themedial globus pallidus (GPm) of a mouse model of dopamine deficiency(SPR-KO), followed by photoinhibition of GPm inputs to VL. As a result,it was confirmed that such photoinhibition reduced rebound firing (seeFIGS. 25 and 26).

The present inventors induced the expression of halorhodopsin in themedial globus pallidus (GPm) of a mouse model of dopamine deficiency(SPR-KO), followed by photoinhibition of GPm inputs to VL. As a result,the locomotion latency and akinesia were recovered and the muscularrigidity and tremor were reduced (see FIGS. 27˜29). Therefore, it wasconfirmed that the GPm inputs could regulate rebound firing of VLneurons in a mouse model of dopamine deficiency (SPR-KO).

The present inventors further confirmed that the spontaneous reboundfiring in WT mice involved in a reduction in motor activity and thepost-inhibitory excitation stabilized ‘standstill’ by inducingsufficient muscle tension. In the meantime, the inventors also confirmedthat the excessive rebound firing in a mouse model of dopaminedeficiency (SPR-KO) could cause pathological conditions that interferewith voluntary motor control, such as akinesia, rigidity, and tremor(see FIGS. 30˜32).

Therefore, rebound firing of VL neurons can be inhibited by irradiatinggreen light after the introduction of a halorhodopsin protein, apolynucleotide encoding the halorhodopsin protein or a vector containingthe polynucleotide above into VL or GPm neurons, by which Parkinson'sdisease can be treated or prevented.

To inhibit rebound firing of VL neurons, a T-type Ca²⁺ channel blockercan be treated to the ventrolateral thalamus (VL) of a subject havingParkinson's disease.

The rebound firing of VL neurons is mediated by the activation of aT-type Ca²⁺ channel. Thus, the rebound firing can be inhibited byinhibiting the T-type Ca²⁺ channel.

In this invention, the “T-type Ca²⁺ channel blocker” indicates asubstance that can selectively inhibit the function of a T-type calciumion channel, which is exemplified by a peptide, a protein, a nucleicacid, a non-peptide compound, a synthetic compound, a fermentationproduct, a cell extract, a plant extract, an animal tissue extract orplasma. These compounds can be novel compounds or well-known compounds.These substances can also include salts.

The salts of the candidate substances herein include salts ofphysiologically acceptable acids (e.g., inorganic acid) or bases (e.g.,organic acid), among which physiologically acceptable acid additionsalts are preferred. For example, salts of inorganic acids (e.g.,hydrochloric acid, phosphoric acid, hydrobromic acid, or sulfuric acid)or organic acids (for example, acetic acid, formic acid, propionic acid,fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,malic acid, oxalic acid, benzoic acid, methanesulfonic acid, orbenzenesulfonic acid) can be used.

The said T-type Ca²⁺ channel blocker can be selected from the groupconsisting of mibefradil, tetramethrin, ethosuximide, SUN-N8075 (DaiichiSuntory Biomedical Research Co Ltd, Japan), efonidipine, Ni²⁺ (divalention of nickel), Y³⁺ (trivalent ion of yttrium), La³⁺ (trivalent ion oflanthanum), Ce³⁺ (trivalent ion of cerium), Nd³⁺ (trivalent ion ofneodymium), Gd³⁺ (trivalent ion of gadolinium), Ho³⁺ (trivalent ion ofholmium), Er³⁺ (trivalent ion of erbium), Yb³⁺ (trivalent ion ofytterbium), U-92032(7-[[4-[bis(4-fluorophenyl)methyl]-1-piperazinyl]methyl]-2-[(2-hydroxyethyl)amino]4-(1-methylethyl)-2,4,6-cycloheptatrien-1-one,Xu and Lee, J. Pharmacol. Exp. Ther., 1994, 268: 1135-1142),penfluridol, fluspirilene, and valproate, but not always limitedthereto. In this invention, ethosuximide is preferably used as theT-type Ca²⁺ channel blocker.

The T-type Ca²⁺ channel blocker can inhibit the expression of a subtypegene that constitutes the T-type Ca²⁺ channel. Particularly, the T-typeCa²⁺ channel blocker above can be an expression inhibitor of one ofthose genes selected from the group consisting of α1G(Ca_(v)3.1),α1H(Ca_(v)3.2), and α1I(Ca_(v)3.3). Herein, the blocker is preferably anexpression inhibitor of Ca_(v)3.1.

The said Ca_(v)3.1 gene can be composed of the nucleotide sequencerepresented by SEQ. ID. NO: 3.

The Ca_(v)3.1 gene expression inhibitor can be any substance capable ofinhibiting the expression or activity of CaV3.1 gene, and can beselected from the group consisting of siRNA, shRNA, and miRNAcomplementarily binding to mRNA of CaV3.1 gene, but not always limitedthereto. Preferably, the said shRNA can be composed of the nucleotidesequence represented by SEQ. ID. NO: 4.

Variants of the nucleotide sequence are included in the scope of thepresent invention. Particularly, a nucleotide sequence having thehomology of at least 70% with the said nucleotide sequence, preferablyat least 80% of homology, more preferably at least 90% of homology, andmost preferably at least 95% of homology with the said nucleotidesequence can be included herein. The “% of sequence homology” with thepolynucleotide is ascertained by comparing the comparison region withtwo optimally aligned sequences. Some of the polynucleotide sequences inthe comparison region can include additions or deletions (i.e., gaps)relative to the reference sequence (without addition or deletion) forthe optimal alignment of the two sequences.

The said siRNA, shRNA, or miRNA can be inserted in a vector. The vectorherein can be selected from the group consisting of a linear DNA vector,a plasmid DNA vector, and a recombinant viral vector. The recombinantviral vector can be selected from the group consisting of retrovirus,adenovirus, adeno-associated virus, and lentivirus. In a preferredembodiment of the present invention, the vector containing siRNA, shRNA,or miRNA is preferably lentivirus, but not always limited thereto.

The vector containing siRNA, shRNA, or miRNA can be introduced in VLneurons by one of those methods selected from the group consisting oftransfection, electroporation, transduction, microinjection, andballistic introduction. According to an embodiment of the presentinvention, transfection is most preferably used, but not always limitedthereto.

In a preferred embodiment of the present invention, GPm inputs werephotostimulated by using the mice lacking the Ca_(v)3.1 gene(Ca_(v)3.1-KO) that encodes the al subunit of T-type Ca²⁺ channels. As aresult, VL neurons showed robust inhibition but significantly diminishedrebound firing, compared with WT neurons, and showed lower correlationswith both motor cortex (M1) and muscular activity (see FIGS. 6 and 7).Therefore, it was confirmed that GPm-VL inhibitory synapses inducedexcitatory motor signals via activating T-type Ca²⁺ channels

The present inventors also investigated the time course of actionpotential firing in VL neurons in response to photostimulation of GPm-VLinputs. As a result, VL neurons evoked a surge of rebound firing within200 ms of the post-inhibitory period. In contrast, Ca_(v)3.1-KO neuronslacked this early-onset rebound firing. Therefore, it was confirmed thatthe early-onset surge of rebound firing depended on the number VLneurons (see FIGS. 8˜10).

The present inventors also confirmed that WT neurons reproduciblyexhibited single spikes soon after the end of hyperpolarization, whilethe early-onset spikes were delayed by blocking T-type Ca²⁺ channelswith Ni²⁺ (see FIGS. 12 and 14). Therefore, it was confirmed that rapidrecovery of membrane potential in VL neurons after inhibition wasdependent on Ca²⁺ influx through the Ca_(v)3.1 channel. It was alsoconfirmed that this rapid recovery facilitated induction of reboundfiring from many VL neurons within a narrow time window (<200 ms), thusyielding a higher excitatory output from the thalamus.

The present inventors also confirmed that significant rebound firing ofVL neurons, decreased locomotor activity and tremor-like behaviors wereobserved in WT mice after photostimulation. In the meantime, reboundfiring of VL neurons and muscular responses were not observed inCa_(v)3.1-KO mice after photostimulation. It was also confirmed thatCa_(v)3.1-KO mice were resistant to the generation of multiple motorabnormalities (see FIGS. 16 and 17). These results strongly suggest thatthe early-onset rebound firing within 200 ms after inhibition wasmediated by Ca_(v)3.1 and acted as the thalamic motor signal.

The present inventors measured the spontaneous rebound firing indopamine-deficient SPR-KO mice showing Parkinson's disease like motorimpairment. As a result, it was confirmed that the dopamine-deficiencyinduced inhibition caused to evoke rebound firing (see FIGS. 23 and 24).

In a preferred embodiment of the present invention, dopamine-deficientSPR-KO mice were treated with ethosuximide. As a result, spontaneousrebound firing of VL neurons was reduced. This result indicated that GPminhibitory input mediated rebound firing in the VL via T-type Ca²⁺channels in a dopamine-deficient state (see FIGS. 26(B) and (C)).

The present inventors treated dopamine-deficient SPR-KO mice withethosuximide. As a result, rebound firing of VL neurons was reduced (seeFIG. 26(C)) and the abnormal motor functions were alleviated (see FIG.29(B)).

In a preferred embodiment of the present invention, the CaV3.1 gene wasknocked-down with shRNA targeted to the dopamine-deficient mouse model(SPR-KO). As a result, rebound firing of VL neurons was reduced and theabnormal motor functions were alleviated (see FIG. 29(C)). Therefore, itwas confirmed that GPm-inputs in the dopamine-deficient mouse model(SPR-KO) mediated rebound firing of VL neurons.

So, rebound firing of VL neurons can be inhibited by treating a T-typeCa²⁺ channel blocker to VL neurons of a subject with Parkinson's diseaseand the inhibition of rebound firing of VL neurons can be efficient intreating or preventing Parkinson's disease.

The halorhodopsin protein, the polynucleotide encoding the halorhodopsinprotein, the vector containing the polynucleotide, or the T-type Ca²⁺channel blocker of the present invention can additionally include anygenerally used carriers, diluents, excipients, or a combination of atleast two of those. The pharmaceutically acceptable carrier can be anycarrier that is able to deliver the active ingredient in human bodywithout limitation, which is exemplified by the compounds described inMerck Index, 13th ed., Merck & Co. Inc., such as saline, sterilizedwater, Ringer's solution, buffered saline, dextrose solution,maltodextrin solution, glycerol, ethanol, and a mixture comprising oneor more of those components. If necessary, a general additive such asantioxidant, buffer, and bacteriostatic agent can be additionally added.The compound of the present invention can be formulated in differentforms including aqueous solutions, suspensions and emulsions forinjection, pills, capsules, granules or tablets by mixing with diluents,dispersing agents, surfactants, binders and lubricants. The compound canfurther be prepared in suitable forms according to ingredients byfollowing the method represented in Remington's Pharmaceutical Science(Mack Publishing Company, Easton Pa., 18th, 1990).

The halorhodopsin protein, the polynucleotide encoding the halorhodopsinprotein, the vector containing the polynucleotide, or the T-type Ca²⁺channel blocker of the present invention can contain one or more activeingredients having the same or similar functions to the above.

The halorhodopsin protein, the polynucleotide encoding the halorhodopsinprotein, the vector containing the polynucleotide, or the T-type Ca²⁺channel blocker of the present invention can be administered orally orparenterally. The parenteral administration includes intracranialinjection, intravenous injection, subcutaneous injection, intramuscularinjection, intraperitoneal injection, and transdermal administration,etc.

The effective dose of the halorhodopsin protein, the polynucleotideencoding the halorhodopsin protein, the vector containing thepolynucleotide, or the T-type Ca²⁺ channel blocker of the presentinvention can be determined according to formulation method,administration method, age, body weight, gender, pathological condition,diet, administration time, administration pathway, bioavailability ofactive ingredients, inactivity rate, concomitant drug, excretion rate,and responsiveness. Particularly, the effective dose of thehalorhodopsin protein, the polynucleotide encoding the halorhodopsinprotein, the vector containing the polynucleotide, or the T-type Ca²⁺channel blocker of the present invention is 0.0001 ng/kg (body weight)to 200 mg/kg (body weight) per day.

The halorhodopsin protein, the polynucleotide encoding the halorhodopsinprotein, the vector containing the polynucleotide, or the T-type Ca²⁺channel blocker of the present invention can be formulated by the methodthat can be performed by those in the art by using a pharmaceuticallyacceptable carrier and/or excipient in the form of unit dose or inmulti-dose container. The formulation can be in the form of solution,suspension or emulsion in oil or water-soluble medium, extract, powder,granule, tablet or capsule. At this time, a dispersing agent or astabilizer can be additionally included.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

The terms and abbreviations used herein have the following meanings.Where the abbreviation is not defined, it can be interpreted as ameaning commonly understood by those in the art.

5HTT-Cre: serotonergic transporter-CreAAV: adeno-associated virusBG: basal gangliaBH4: tetrahydrobiopterinEF1α: elongation factor 1αEMG: electromyographyERP: event-related potentialsETX: ethosuximideGPm: medial globus pallidushChR2: channelrhodopsin-2IPSP: inhibitory postsynaptic potentialsKO: knockoutLFP: local field potentialsLGN: lateral geniculate nucleusM1: motor cortexMLR: mesencephalic locomotor regionMUA: multi-unit activitynRT: reticular thalamic nucleiPD: Parkinson's diseasePPN: pedunculopontine nucleusSNr: substantia nigra pars reticulataSPR-KO: sepiapterin reductase knockoutSTN: subthalamic nucleiSUA: single-unit activityVL: ventrolateral thalamusWT: wild typeZI: zona incerta

Example 1: Construction of hChR2 Expressing Mouse

Mice (WT, C57BL/6J, n=31) over 8 weeks old were used. Animal care andhandling were performed according to the guidelines of the Animal Careand Use Committee of the Korea Advanced Institute of Science andTechnology (KAIST, Korea). WT littermates were generated by matingheterozygous mutants (C57BL/6J background). SNr(substantia nigra parsreticulata) or GPm(medial globus pallidus) neurons was infected with anadeno-associated virus (AAV) vector harboring the gene for thelight-activated cation channel channelrhodopsin-2 (hChR2) driven by theelongation factor 1α (EF1α) promoter (FIG. 1). TheAAV2/1-EF1α-DIO-hChR2(H134R)-mCherry-WPRE (Cat #AV-1-20297P; titer:5.36×10¹² gc/ml) was generated by the Vector Core Facility at theUniversity of Pennsylvania (USA). For virus injection, all mice wereanesthetized with avertin (20 mg/ml of tribromoethanol, 20 μl/g i.p.)and placed in a stereotaxic apparatus (David Kopf Instruments, USA). Allinjections were delivered at a rate of 0.1 μl/min. For expression ofopsins in a limited target area, AAV9-CMV-CRE-EGFP andAAV2/1-EF1α-DIO-hChR2(H134R)-mCherry-WPRE was mixed at a 1:1 ratio. Atotal of 1.0 μl of virus mixture was injected into the GPm (−1.3 mmanteroposterior [AP]; −1.8 mediolateral [ML]; 4.25 dorsoventral [DV]).For expression of opsins in SNr (−3.3 mm AP; −1.4 ML; 4.9 DV), nRT (−0.7mm AP; −1.3 ML; 3.5 DV) and ZI (−2.5 mm AP; −1.85 ML; 3.8 DV), the virusprepared in same titer was used with different volumes (0.8 ul, 0.4 ul,and 0.5 ul, respectively) according to the size of target area.

After injection, a fiberoptic probe with an external metal ferrule (200μm diameter, 0.39 NA; Doric Lenses Inc., Canada) was implanted into theVL thalamus and fixed to the skull using Super-Bond (Sun Medical Co.,Japan) and dental cement (Stoelting, USA). Coordinates for the VLthalamus were −1.0 mm AP, −1.1 ML, and 3.4 DV.

Example 2: Construction of Ca_(v)3.1-KO Mouse

The Ca_(v)3.1-KO mice of the present invention were the offsprings ofthe Ca_(v)3.1-KO mouse prepared according to the method described inLack of the burst firing of thalamocortical relay neurons and resistanceto absence seizures in mice lacking alpha(1G) T-type Ca²⁺ channels (Kimet al., Neuron, 31: 35-45, 2011). The specific procedure for preparingthe Ca_(v)3.1-KO mouse described in the literature is as follows.

A mouse cDNA of the Ca_(v)3.1 gene (cacna1G) sequence was isolated byRT-PCR and was used for isolating mouse genomic DNA clones containingthe Ca_(v)3.1 locus from a phage library. The targeting vectorcontaining 11.7 kb homologous fragments with double selection markers,neo and TK, was made and introduced into J1 embryonic stem cell lines.The targeted ES clones were identified by Southern blot analysis andused in the generation of germline chimeras, as previously described(Kim et al., 1997). Male germline chimeras were crossed with femaleC57BL/6J mice to obtain F1 heterozygotes (Ca_(v)3.1+/−) and these F1were intercrossed to obtain homozygous mutant mice (Ca_(v)3.1−/−).

Example 3: Construction of Halorhodopsin Expressing WT Mouse

To design an optogenetic experiment, Halorhodopsin (eNpHR3.0), alight-dependent chloride pump, was expressed in WT mice to facilitatephotoinhibition of VL neurons while hChR2 was expressed in GPm to allowphotostimulation of GPm inhibitory inputs to the VL.

The AAV2/9-EF1α-DIO-eNpHR3.0-EYFP-WPRE (Cat #AV-9-26966P) was generatedby the Vector Core Facility at the University of Pennsylvania (USA). Forvirus injection, all mice were anesthetized with avertin and placed in astereotaxic apparatus. All injections were delivered at a rate of 0.1μl/min. For expression of opsins in a limited target area,AAV9-CMV-CRE-EGFP and AAV2/9-EF1α-DIO-eNpHR3.0-EYFP-WPRE was mixed at a1:1 ratio. A total of 1.0 μL of virus mixture was injected into the VL.Coordinates for the VL thalamus were −1.0 mm AP, −1.1 ML, and 3.4 DV.

After injection, a fiberoptic probe with an external metal ferrule wasimplanted into the VL thalamus and fixed to the skull using Super-Bondand dental cement. Coordinates for the VL thalamus were −1.0 mm AP, −1.1ML, and 3.4 DV.

Histological analysis revealed that ˜50% of thalamic neurons expresseNpHR 3.0 (FIG. 19).

Example 4: Construction of SPR-KO Mouse

The SPR-KO mice of the present invention were the offsprings of theSPR-KO mouse prepared according to the method described in A murinemodel for human sepiapterin-reductase deficiency (S. Yang et al., Am. J.Hum. Genet., 2006). The specific procedure for preparing the SPR-KOmouse described in the literature is as follows.

The mouse Spr gene encodes 261 aa and consists of three exons.Previously a phage clone containing the entire Spr gene from a 129/SvJ(129) mouse genomic DNA library was isolated. To construct a targetingvector, a 3.3-kb HindIII-SacI fragment including a part of exon 1 and a3.8-kb SacI-HindIII fragment containing the exon 3 region weresequentially inserted into the XhoI and XbaI sites, respectively, of thepPNT vector. The phosphoglycerate kinase (PGK)-neomycin cassettereplaced a portion of exon 1, intron 1, and the entire exon 2 encodingthe short-chain dehydrogenase/reductase domain. After electroporation ofthe linearized construct, G418-resistant and1-(2-deoxy-2-fluoro-1-β-d-arabinofuranosyl)-5-iodouracil(FIAU)-resistant colonies were selected. Approximately 300double-resistant colonies were screened by Southern-blot analysis usinga 5′ external probe. One of the three positive clones was microinjectedinto C57BL/6J (B6) blastocysts to generate chimeras, which were crossedwith B6 mice to establish and maintain the Spr+/− mouse line on a mixed129/B6 hybrid background. The genotype of offspring from the breeding ofheterozygous mice was determined with PCR primer sets SprF1 (SEQ. ID.NO: 5; 5′-AAGTGGTGCTGGCAGCCGCCGAT-3′) and NeoP3 (SEQ. ID. NO: 6;5′-CGGTGCTGTCCATCTGCACGAGAC-3′), for detection of the mutant allele, andsrex2F (SEQ. ID. NO: 7; 5′-CCTCCATGCTCTGTTTGACT-3′) and srex2R (SEQ. ID.NO: 8; 5′-GTTCCCCTCCTTGCCTAGC-3′), for detection of the wild-typeallele. The genomic region amplified by the srex2F and srex2R primer setwas deleted in the mutant allele, and thus no PCR amplification occursfor the Spr−/− mice.

The survival of SPR-KO mice was maintained by treating daily with BH4(Schircks Laboratories, Switzerland) in ascorbic acid (Sigma, USA)beginning on P2; N-acetyl-L-cysteine solution (Sigma) was used as avehicle control. The dose of BH4 and vehicle are based on the method ofYang et al. (2006).

Example 5: Construction of Halorhodopsin Expressing SPR-KO Mouse

A SPR-KO mouse expressing halorhodopsin was constructed by injecting avirus expressing halorhodopsin into GPm according to the methoddescribed in Example 3 to the SPR-KO mouse constructed in Example 4.

GPm was infected with an adeno-associated virus (AAV) harboring eNpHR3.0under the control of the EF1a promoter. Illumination with green light(532 nm) was used to photoinhibit GPm inputs, and multi-unit recordingswere used to determine the effects of photoinhibition (FIG. 25).

Example 6: Construction of SPR-KO Mouse Expressing shRNA TargetingCa_(v)3.1 Gene

The SPR-KO mouse prepared in Example 4 was injected with a virusexpressing shRNA targeting CaV3.1 gene prepared according to thefollowing method.

A lentiviral vector expressing short hairpin RNA (shRNA) to target theCa_(v)3.1 T-type calcium channel was constructed as the paper by Kim etal. (2011). The recombinant lentiviral vectors were produced andconcentrated commercially (Macrogen LentiVector Institute). Thelentivirus titers of 2×10⁶ transduction units/ml were used. The solutioncontaining the viruses carrying the Ca_(v)3.1-shRNA or the scrambledcontrol were injected into the right VL thalamus with Nanofil 33G bluntneedles and a Nanofil syringe (World Precision Instruments) using amicro syringe pump (Eicom). 10 days after viral transduction, analyseswere performed.

Experimental Example 1: Photoactivation of the GPm-VL Pathway ModulatesLocomotion

The effect of basal ganglia (BG) inhibitory input on the thalamus wasexamined.

In Example 1, GPm (medial globus pallidus), SNr (substantia nigra parsreticulata), ZI (zona incerta), or nRT (reticular thalamic nucleus)neurons were infected with an adeno-associated virus (AAV) vectorharboring the gene for the light-activated cation channelchannelrhodopsin-2 (hChR2) driven by the elongation factor 1α (EF1α)promoter (FIG. 1).

To determine the specific role of GPm-VL inputs, the axons of GPmneurons were photostimulated by illuminating (473 nm) the core area ofthe VL. For optogenetic stimulation experiments, 473 nm light wasprovided by a diode-pumped solid-state laser (CrystaLaser, USA)controlled by a pulse generator (Agilent, USA).

For analyses of fine movement and locomotion, mice were subjected toboth cylindrical- and square chamber assays. First, mice were placed ina cylindrical glass chamber (diameter, 15; height, 20 cm) and allowed toexplore the chamber and adjust to the fiber-optic cable for ˜1-3 min.Light stimuli (20 Hz, 5-ms pulses for 473 nm) were applied in 3-5sessions, with each session lasting for 15-20 s. After the sessions,mice were placed directly in an acrylic chamber (25×28×22 cm) andallowed to explore for 1-3 min, and the same light stimulation sessionswere repeated. The assay was performed between 1:00 PM and 6:00 PM in adimly lit room and recorded with a camcorder at a sampling rate of 25samples/s. The video recordings were analyzed using EthoVision XT 8.5(Noldus Information Technology).

Compared to mice infected with control virus expressing mCherry only,hChR2 mice displayed significantly less spontaneous locomotor activityin response to VL photostimulation, indicating hypokinesia (FIG. 2). Incontrast, photostimulation of VL inputs from SNr and other inhibitoryinputs from the nRT or ZI neurons had no significant effects onlocomotion (not shown). These results clearly indicate that the GPm-VLcircuit is critical for the suppression of locomotion and plays a uniquerole compared to other inhibitory inputs to VL.

Experimental Example 2: Photoactivation of the GPm-VL Pathway InducesMotor Responses Through Muscular Contraction

The present inventors examined whether the GPm-VL circuit affects thegeneration of motor signals. The electromyography (EMG) was applied tomeasure muscular activity (FIG. 1C).

<2-1> In Vivo Spike Recording in a Waking State

For implanting head-plate and EMG electrodes, WT mice were anesthetizedwith avertin and placed in a stereotaxic device. A custom-designedhead-plate with a circular window (1 cm diameter) was implanted bycementing to the skull with Super-Bond and dental cement. For recordingmuscle activity of the forelimb, an EMG electrode was bilaterallyimplanted in the lateral part of the triceps brachii muscle. Forrecording muscle activity of neck muscles, two Teflon-coated tungstenEMG electrodes (A-M Systems Inc., USA) were implanted in the neck. Allconnectors were fixed to the head-plate with dental cement. Allelectrodes for EMG recordings were then connected to an electrodeinterface board (EIB-16; Neuralynx).

After at least a 3-d recovery period, mice were habituated for 15 min tothe head-restrained conditions used for recording sessions, andrecordings were performed the next day.

Three trials of 10 s optical stimulation, with a 50 s inter-trialinterval, were performed. Three-trial sets of optical stimulation wererepeated using the following conditions: 5-, 25-, 50- and 100-ms pulsesat 1 and 5 Hz, and 5- and 25-ms pulses at 20 Hz. EMG was recorded with aDigital Lynx acquisition system (Neuralynx). Data were digitized at 30-5kHz for EMGs.

To identify the activated EMG signals, EMG spikes above a threshold(2.5-3.5×SD of basal EMG amplitude) were extracted from raw EMG signalsusing Spike Extractor software (Neuralynx). Peri-event histograms atvarious frequencies were calculated using Neuroexplorer (NexTechnologies). For the frequency analysis of 20 Hzphotostimulation-induced EMG activities, the power spectrogram wascomputed from rectified EMG signals using Neuroexplorer software.Representative traces in each frequency range were computed using theButterworth filter in MATLAB (version R2013a; Mathworks, USA) software.

Shortly after photostimulation of GPm-VL inputs, muscle contractionsoccurred. The magnitude of this activity varied with the frequency ofphotostimulation (FIG. 3) and light pulse duration (not shown).Photostimuli at 1 Hz reliably evoked muscle twitches (FIG. 3). Incontrast, low-frequency (5 Hz) trains of light flashes inducedtremor-like activity at the same frequency (green arrowheads in FIG. 3)whereas high-frequency trains (20 Hz)—mimicking the frequency of GPmoscillations observed in PD—induced both high-frequency muscle activity(red arrowheads in FIG. 3, yellow EMG signals in FIG. 4) andlow-frequency tremor activity (green arrowhead in FIG. 3, red and greenEMG signals in FIG. 4).

<2-2> Rigidity Test

The high-frequency muscle activity may reflect muscular rigidity, astate of continuous contraction. To test the degree of rigidity, theability of mice to hold onto a bar (FIG. 5) was examined.

The front paws of a mouse were positioned on a 2-mm horizontal wire bar4 cm above the ground, and the latency to paw release was analyzed.Three trials were performed for each mouse, with each trial lasting for1 min; the results for each mouse are presented as the average of thethree trials. The test was performed between 12:00 PM and 6:00 PM.

During 20 Hz photostimulation, the latency of spontaneous bar releasewas significantly delayed compared to the performance observed withoutphotostimulation, indicating rigidity (FIG. 5). These results signifythat GPm inhibitory input to VL alone is sufficient to trigger signalsfor muscle contraction, potentially leading to various motor responses,including suppressed locomotor activity, tremor, and rigidity.

Experimental Example 3: GPm-VL Inputs Induce Inhibition and ReboundFiring in VL Neurons by Activating T-Type Ca²⁺ Channels <3-1>Correlation of Neural and Motor Responses in WT Mice

To identify the neural correlates of these motor responses, the activityof neurons in VL and motor cortex (M1), as well as muscle activityduring photostimulation of the GPm-VL input were monitored (FIG. 6(A)).

VL multi-unit activity, EMGs, and cortical LFPs were recordedsimultaneously in vivo following optical stimulation. For implantinghead-plate and EMG electrodes, the electrodes were implanted accordingto the method described in Example <2-1>. For recording cortical LFPs, atungsten wire (Cat. #796000; A-M Systems Inc.) was acutely implantedinto the M1 cortex (0 mm AP, −1.0 ML, 1.0-1.5 DV) during preparation forthe recording session. All electrodes for LFP and EMG recordings werethen connected to an electrode interface board (EIB-16; Neuralynx).

After at least a 3-d recovery period, mice were habituated for 15 min tothe head-restrained conditions used for recording sessions, andrecordings were performed the next day. Mice were prepared forrecordings by anesthetizing with isoflurane (1.5% in oxygen), afterwhich their head-plate was fixed to a holder device. Holes were drilledin the skull above the right VL for multi-unit activity (1.0 mm AP, −1.0ML), the M1 for LFPs (0 mm AP, −1.0 ML) and the temporal cortex for theground electrode (2.0 mm AP, 2.0 ML); the dura was cleanly removed toallow insertion of electrodes. An optrode for recording VL multi-unitactivity was fixed to a micromanipulator (Stoelting) and lowered intothe VL thalamus. LFP and ground electrodes were localized in the M1 andtemporal cortex, respectively, and fixed to the head-plate withcyano-acrylate (Loctite; Henkel, Germany). The holes were sealed with1.5% liquid agar, and the mice were allowed to recover from theanesthesia. Recording sessions were started after the mice fullyregained consciousness. VL neurons connected to the GPm were identifiedby delivering 50-ms-width light pulses at 1 Hz and observing VL neuralactivity every 100-150 mm in the VL region.

After light-responsive VL neurons were detected, basal neural activitywas recorded for 5-10 min, and then three trials of 10 s opticalstimulation, with a 50 s inter-trial interval, were performed.Three-trial sets of optical stimulation were repeated using thefollowing conditions: 5-, 25-, 50- and 100-ms pulses at 1 and 5 Hz, and5- and 25-ms pulses at 20 Hz. After each trial, the optrode was lowered200-500 μm to detect another light-responsive VL neuron. When neuronalfiring was detected, basal activity was recorded and opticalstimulations were repeated. Upon completion of recordings, mice weresacrificed after making an electrolytic lesion (1 mA, 5 s) to confirmthe anatomical location. Neural signals, including VL multi-unitactivity, M1 LFPs and EMGs, were recorded with a Digital Lynxacquisition system (Neuralynx). Data were digitized at 32 kHz andband-pass filtered at 300-5 kHz for multi-unit activity, at 0.5-50 Hzfor LFPs, and at 30-5 kHz for EMGs. TTL signals from the pulse generatorwere recorded concurrently with neural signals.

The light-induced neural activities were computed as the perieventfiring rate histogram in 25-ms bins. To analyze the rebound firing(post-inhibitory activities) induced by photostimuli, the subset ofmulti- and single-units which showed both significant silencing wasutilized during the light illuminations and firing increase over thenthe basal activity after the photostimuli. All analyses were performedusing MATLAB (Mathworks, USA) and Neuroexplorer (Nex Technologies, USA)software.

Activation of GPm-VL inputs (blue bars in FIG. 6(A)) led to the expectedinhibition of VL neurons during light flashes (FIG. 6(A)). However, atthe end of each light flash, VL neurons showed a surge of actionpotentials (VL in FIG. 6(A)). This rebound firing was accompanied byincreased activity in the motor cortex (M1), as evident from local fieldpotentials (LFP in FIG. 6(A)), and muscle responses, indicated byenhanced EMG activity in neck and arm muscles (Neck-EMG and Arm-EMG inFIG. 6(A)). These results support the possibility that activation ofGPm-VL inhibitory synapses induces rebound firing of VL neurons, which,in turn, stimulates the motor cortex and causes muscle contraction.

<3-2> Correlation of Neural and Motor Responses in Ca_(v)3.1-KO Mice

To determine the molecular basis underlying the rebound firing producedin VL neurons by GPm inputs, the same experiment as described in Example<3-1> was performed using the Ca_(v)3.1-KO mice constructed in Example2. The Ca_(v)3.1 gene encodes the al subunit of T-type Ca²⁺ channelsknown to be critical for inducing rebound burst firing in response toinhibition.

When GPm inputs were photostimulated in vivo, Ca_(v)3.1-KO neuronsshowed robust inhibition (FIG. 7) but significantly diminished reboundfiring, compared with WT neurons (FIG. 6(B)). Remarkably, absolutely nomuscular responses were observed (FIG. 6(B)) under variousphotostimulation conditions. The activity of VL neurons in Ca_(v)3.1-KOmice consistently showed lower correlations with both M1 and muscularactivity, relative to WT neurons (FIG. 6). Based on these findings,GPm-VL inhibitory synapses induce excitatory motor signals viaactivating T-type Ca²⁺ channels.

Experimental Example 4: The Motor Signal Depends on the Number of VLNeurons that Generate Rebound Firing in Response to Inhibition

To gain further insight into how VL neurons induce motor signals inresponse to GPm inhibitory input, the time course of action potentialfiring in VL neurons from WT and Ca_(v)3.1 KO mice was analyzed inresponse to photostimulation of GPm input.

The activity of VL neurons was measured according to the VL multi-unitactivity test method described in Experimental Example <3-1>.

For single-unit analysis, spikes were extracted using Spike Extractorsoftware (Neuralynx), then clustered semi-automatically using theAutoKlustaKwik function in SpikeSort 3D software (Neuralynx), followedby manual adjustment of the clusters. Only high-quality single unitswere used for data analysis. This protocol was also used in the other invivo recording experiments. Low-threshold spike (LTS) bursts weredefined using the criteria of Kraus et al. (2010), as follows: 1) apre-silent period >100 ms, 2) interspike intervals <4 ms following thefirst two spikes, and 3)<4-ms interspike intervals between subsequentspikes. All other spikes were considered tonic spikes. After clusteringsingle units, all spikes in each unit were sorted into tonic spikes orLTS bursts.

WT neurons evoked a surge of rebound firing within 200 ms of thepost-inhibitory period that exceeded the firing rate measured prior toinhibition in multi-units (FIGS. 8(A) and 8(C)). In contrast,Ca_(v)3.1-KO neurons lacked this early-onset rebound firing and slowlyregained baseline activity more than 200 ms after photostimulation(p<0.05, FIGS. 8(B) and 8(C)).

It was initially considered that possibility that the temporal patternof spiking or excitability of individual VL neurons could explain themotor response evoked by activation of BG inputs.

One candidate mechanism is low-threshold burst firing, which consist ofmultiple action potentials at 200-400 Hz that serve as a strong motorsignal. Ca_(v)3.1-KO VL neurons lack low-threshold burst spikes (notshown) and these mice do not show photostimulation-induced motorabnormalities (FIGS. 6(B) and 7). However, single-unit recordingsrevealed that low-threshold burst spikes were rare during the reboundfiring induced by various photostimulation conditions in WT mice (notshown). In addition, in WT mice most motor responses were associatedwith rebound firing that lacked low-threshold burst spikes (not shown).This indicates that low-threshold burst spikes are not involved in thegeneration of motor signals during GPm-VL photostimulation.

Next, the possibility that the firing rate of WT neuron is higher thanKO neuron in the post-inhibitory period was tested. These data showed nosignificant differences in the peak firing rate (FIG. 8(D), (E), (F) andFIG. 9) or in the average firing rates of individual VL neurons betweenthe two genotypes in the post-inhibitory period (not shown). Instead, asindividual WT neurons reached a peak firing rate faster than KO neurons(LP50 for WT=187 ms; LP50 for KO=362 ms), a greater portion of neuronsshow peak firing within 200 ms of the post-inhibitory period (78% for WTvs. 14% for KO) (FIG. 10). These results suggest that the early-onsetsurge of rebound excitability, which might be critical for motorresponse induced by GPm-VL photostimulation, seems to depend on thenumber VL neurons with rebound firing rather than low-threshold burstspikes or averaged firing rate of individual neurons.

Experimental Example 5: T-Type Ca²⁺ Channels Facilitate RapidRepolarization of Membrane Potentials to Generate Early-Onset Firing

To obtain detailed mechanistic information on how individual VL neuronsproduce early-onset rebound firing independently from low-thresholdburst spikes, whole-cell patch-clamp recordings in VL slices from WT andCa_(v)3.1-KO mice were performed to obtain high-resolution measurementsof electrical activity in single neurons (FIG. 11).

<5-1> Whole-Cell Patch-Clamp Recording to Measure Neuronal Responses toPhotostimuli

Brain slices were prepared from WT and Ca_(v)3.1-KO mice 2 weeks to 7months after the injection of AAV into the medial globus pallidus (GPm)region. In brief, isolated brains in high-sucrose artificialcerebrospinal fluid (sACSF; in mM: 87 sodium chloride, 75 sucrose, 25NaHCO₃, 2.5 KCl, 0.5 CaCl₂), 7 MgCl₂, 1.25 NaH₂PO₄ and 25 d(+)-glucose),maintained at pH 7.4 by gassing with 95% O₂/5% CO₂, were sliced into350-400-mm-thick coronal sections using a Vibratome (VT-1200; Leica,Germany). Slices were transferred to an incubation chamber filled withoxygenated ACSF consisting of (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄,25 NaHCO₃, 25 d (+)-glucose, 2 CaCl₂), 2 MgCl₂, 3 sodium pyruvate, and 1ascorbic acid. Slices were incubated at 36° C. for 30 min and weremaintained at room temperature for at least 30 min prior to use.

Whole-cell patch-clamp recordings were performed at 32° C. under anupright microscope (FV1000MPE, Olympus, Japan) in a recording chamberperfused with 95% O₂/5% CO₂-aerated ACSF as the extracellular solution.VL thalamus neurons were identified using infrared differentialinterference contrast (IR-DIC) optics in combination with a digitalvideo camera (MCE-B013-U; Mightex, Canada). Whole-cell patch-clamprecordings were obtained from these neurons using glass pipettes (5-12MΩ) filled with an internal solution consisting of (in mM) 130K-gluconate, 2 NaCl, 4 MgCl₂, 20 HEPES, 4 Na₂ATP, 0.4 Na₃GTP, 0.5 EGTA,and 10 Na₂ phosphocreatine. The osmolarity of the solution was 290-295mOsm, and the pH was adjusted to 7.25 using 1 M KOH. Unless otherwiseindicated, all current measurements were made using a holding potentialof −60 mV.

Electrical responses were acquired with a patch-clamp amplifier(Multiclamp 700B; Molecular Devices, USA) and pClamp software (MolecularDevices), digitized at 20 kHz using an A-D converter (Digidata 1440A;Molecular Devices), and analyzed using Clampfit (Molecular Devices). Inparallel with the patch-clamp recordings, photostimuli were appliedthrough a 25×(1.05 NA) water-immersion objective lens; the entire widthof the microscope field (500 μm diameter) was illuminated. A mercury arclamp (USH-1030L; Olympus), used to provide light to activate ChR2, wasfiltered using a band-pass filter (465-495 nm). Light pulses (5 mW/mm²,10-ms duration) were applied at 20 Hz for 10 s, controlled by anelectronic shutter (Uniblitz VS25; Vincent, USA).

First, the properties of GPm-to-VL synaptic transmission wereinvestigated. Photostimulation of GPm input induced inhibitorypostsynaptic potentials (IPSP) in VL neurons (FIG. 11(A)). Nosignificant differences between WT and Ca_(v)3.1-KO VL neurons wereobserved in terms of mean IPSP amplitude (FIG. 11(C)) or the fraction ofVL neurons exhibiting IPSPs in response to photostimulation (FIG.11(B)). These results indicate that GPm-VL inhibitory synaptictransmission is not altered in Ca_(v)3.1-KO mice.

<5-2> Whole-Cell Patch-Clamp Recording to Observe Rebound Firing inThalamic Neurons

Next, the intrinsic firing properties of VL neurons from WT andCa_(v)3.1-KO mice were examined.

Adult male mice (7-19 weeks old) were anesthetized by i.p. injection ofavertin, and sacrificed. The isolated brains were immersed in ice-coldartificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl, 1.25NaH₂PO₄, 25 NaHCO₃, 25 dextrose, 2 CaCl₂), 2 MgCl₂, 3 Na-pyruvate, 1ascorbic acid, maintained at pH 7.4 by gassing with 95% O₂/5% CO₂) andwere sliced into 250 μm thick coronal sections using a Vibratome. Theslices were transferred to an incubation chamber filled with NMDGrecovery solution (in mM: 92 NMDG, 92 HCl, 30 NaHCO₃, 2.5 KCl, 0.5CaCl₂), 10 MgSO₄, 1.2 NaH₂PO₄, 20 HEPES, 5 Na-ascorbate, 3 Na-pyruvate,2 thiourea, 25 dextrose, maintained at pH 7.4 by gassing with 95% O₂/5%CO₂) for 15 min at 36° C. Then the slices were maintained in oxygenatedACSF at room temperature for at least 1 hr prior to use.

VL thalamus neurons were recorded in oxygenated ACSF at 28-30° C. andvisualized under an upright microscope (BX-51WI; Olympus, Japan).Whole-cell patch clamp recordings were performed using glass pipettes(3-4 MS)) filled with internal solution (in mM: 130 K-gluconate, 2 NaCl,4 MgCl₂, 20 HEPES, 4 Na₂ATP, 0.4 Na₃GTP, 0.5 EGTA, pH 7.25, 290-295mOsm). VL thalamus neurons were ruptured at −70 mv. IT was identified involtage-clamp mode using −30 mV hyperpolarization steps (200 ms) from aholding potential of −70 mV, then 5 mV depolarization steps (500 ms)from the holding potential. Spike latency and jitter afterhyperpolarization were tested in current-clamp mode. After establishingthe smallest holding current (maximum, 600 pA) at which the cellspontaneously and stably fired (1-8 Hz), 100 ms hyperpolarizationcurrents, which varied from cell to cell (−215 to −400 pA) were applied,until a single rebound spike appeared after hyperpolarization.

T-type calcium channels were blocked by applying 500 μM NiCl₂ (Sigma) inACSF for at least 10 min. After confirmed the effect of NiCl₂application by recording IT, holding currents at which the cellspontaneously and stably fired (maximum, 550 pA) were identified asdescribed above. From these holding currents, we applied the same amountof 100 ms hyperpolarizing current as was applied prior to NiCl₂treatment.

To confirm the effect of T-type calcium channel on rebound firing, theexperiment with Ca_(v)3.1 KO mice was performed. After establishing thesmallest holding current at which the cell spontaneously and stablyfired (1-8 Hz), we applied 100 ms, −300 pA hyperpolarization currentswere applied to VL thalamus neuron of Ca_(v)3.1 KO mice (Ra; 15.64±1.57Mohm, holding; −52.04±11.19 pA) and wild-type mice (Ra; 14.25±3.23 Mohm,holding; −74.78±21.96 pA).

To mimic GPm-mediated inhibition in vivo, a hyperpolarizing currentpulse (100 ms duration) was injected into VL neurons at a restingmembrane potential of approximately −55 to −60 mV (FIGS. 12(A) and (B)).In every trial, WT neurons reproducibly exhibited single spikes soonafter (˜70 ms) the end of hyperpolarization. The timing of theseearly-onset spikes was replicated in many individual neurons, even inrecordings obtained from different thalamic slices (FIGS. 12(C) and(D)). Blocking T-type Ca²⁺ channels with nickel (Ni²⁺, not shown) didnot significantly alter neuronal intrinsic properties or firing rate(not shown). However, Ni²⁺ treatment did produce a delay in early-onsetspikes (FIG. 13(A)), which became much more irregular (FIG. 13(C)). As aresult, rebound spikes occurred within a narrow time window (<200 ms)after application of the hyperpolarizing inhibitory stimulus in WTneurons but were delayed and dispersed in Ni²⁺-treated neurons (FIG.14). Ni²⁺ treatment recapitulated the differences in post-inhibitoryfiring activity between WT and Ca_(v)3.1-KO neurons observed in vivo(compare to FIG. 10). Additionally, similar delays in the timing ofearly-onset spikes were observed in KO neurons (FIGS. 13(B) and (D)).

The delay in onset of post-inhibitory spikes caused by Ni²⁺ (FIGS. 12,13 and 14) may arise from delayed recovery of membrane potential afterinhibition. This recovery rate determines the timing of action potentialfiring and is dependent on T-type Ca²⁺ channel activity, because it wasslowed by Ni²⁺ (FIG. 15). Thus, in VL neurons rapid recovery frominhibition depends on Ca²⁺ influx through the Ca_(v)3.1 channel.Furthermore, this recovery facilitates induction of rebound firing frommany VL neurons within a narrow time window, thus yielding a higherexcitatory output from the thalamus.

Experimental Example 6: Early-Onset Surge of Rebound Firing MediatesMotor Dysfunction <6-1> Correlation of Rebound Firing and MotorAbnormalities in CorWT and CaV3.1-KO Mice

To define the causal relationship between rebound firing and motorabnormalities, first, the analyzed event-related potentials (ERP) wasanalyzed. Neural and muscular responses of WT and Ca_(v)3.1-KO mice weremeasured according to the EMG Recordings and VL single-unit activitymethod described in Experimental Example <3-1>. After the measurement,rebound firing analysis was performed as follows.

The rebound firing was identified from total multi-unit activitiesextracted from the spikes in each tetrode using threshold-based SpikeExtractor software (Neuralynx). The onset time for rebound firing wasidentified as the time when the firing rate was increased over the meanfiring rate, after the inhibitory period (over 50-ms) which shows lowerfiring rate than the mean. Because the reliable mean firing rate couldnot be computed within short duration, alternative method was utilizedin the recording dataset shorter than 30 s. The 50-ms window was shiftedevery 10 ms and the firing rate was computed in each window. In caseswhere the firing rate steadily increased in three consecutive windows,the starting point of the second window was defined as the point atwhich firing rate increased. Since the firing rates in the first windowwere lower than basal activity, this method successfully identify thepost-inhibitory activities.

All neural and muscular responses to each photostimulus (5, 25 or 50 mspulse) were averaged, thereby filtering out random and uncorrelatedsignals (FIG. 16).

In response to brief photostimuli (<5 ms), WT mice exhibited a robustreduction in the firing rate of VL neurons but failed to show reboundfiring or muscular responses (FIG. 16(A)). In contrast, flashes 25 ms orlonger efficiently induced muscular responses (blue arrows in FIG. 16)accompanied by VL neuron inhibition and a greater amount of reboundfiring that peaked approximately 170 ms after application of thephotostimulus (red arrowheads in FIG. 16).

In Ca_(v)3.1 KO mice, photostimulation of GPm-VL synapses reduced thefiring rate during the stimulus but did not induce significant reboundfiring or muscular responses (asterisks in FIG. 16(B)).

Additionally, behavioral responses during photostimulation of GPm-VLsynapses between WT and Ca_(v)3.1 KO mice were compared. Locomotoractivity test was performed according to the method described inExperimental Example 1, and tremor test was performed according to theprocedure described below. Each trial lasted for 30 min, and all trialswere video-recorded. The test was performed between 12:00 PM and 6:00PM. The intensity of tremor was scored by two independent investigatorsblinded to group-identifying information. Tremor intensity was rated ona scale of 0 to 4, as described by Lars M. Ittner et al. (Ittner et al.,2008), where 0 indicates no tremor, 1 intermittent slight tremor, 2intermittent tremor, 3 strong tremor with rare quiescent periods, and 4continuous tremor.

While WT mice showed decreased locomotor activity and tremor-likebehaviors, as illustrated in FIGS. 1 to 5, Ca_(v)3.1-KO mice wereresistant to the generation of multiple motor abnormalities (FIG. 17).These data strongly suggest that early-onset rebound firing within 200ms after inhibition, mediated by Ca_(v)3.1, functions as the thalamicmotor signal.

<6-2> Relationship Between Rebound Firing and Motor Abnormalities ofMice Expressing Halorhodopsin in VL Neurons and hChR2 in GPm

To further test this suggestion, an optogenetic experiment in whichrebound firing was inhibited during specific time windows was designed(FIG. 18). For this purpose, mice expressing halorhodopsin (eNpHR3.0)were constructed in Experimental Example 3. HChR2 was expressed in GPmto allow photostimulation of GPm inhibitory inputs to the VL, whilehalorhodopsin could photoinhibit VL neurons.

With this arrangement, 473 nm light (blue rectangles in FIG. 20) wasapplied to activate GPm-VL inputs and 561 nm light (green rectangles inFIG. 20) was used to inhibit postsynaptic activity during either theearly or late post-inhibitory period (FIG. 20). FIG. 20 illustrates therepresentative EMG responses measured according to the method of EMGRecordings described in Experimental Example <3-1>. Whilephotoactivation of GPm-VL inputs robustly induced muscular contractions(FIG. 20(A)), this motor response was abolished by postsynapticphotoinhibition during the early (<200 ms) post-inhibitory period (FIG.20(B) and FIG. 21).

In contrast, photoinhibition >200 ms after activation of GPm input hadlittle effect, allowing robust motor responses similar to controls (FIG.20(C) and FIG. 21). These results suggest that the motor signal inducedby the GPm input depends on very short-term integration of firing,rather than long-term integration or the average firing rate. Thus, thenumber of neurons that evoke rebound firing with similar timing withinthe first 200 ms post-inhibition controls the amount of excitatoryoutput from the VL.

It was further investigated whether rebound firing mediates thehypokinesia produced by activating GPm inputs to VL. As in ExperimentalExample <6-1>, locomotor activity test and tremor test were alsoperformed with mice expressing hChR2 in GPm and halorhodopsin in VLneurons.

Rhythmic photostimulation of the inputs via blue light (473 nm) induceda significant reduction in locomotion (FIG. 22(A)) similar to theresults shown in FIG. 2. However, pairing photostimulation of GPminhibitory inputs with photoinhibition of VL neurons via green light(561 nm) efficiently restored locomotion activity (FIG. 22(B)). Theseresults strongly support the conclusion that multiple motor dysfunctionsarise from rebound firing of VL neurons.

Experimental Example 7: Dietary Supplementation Rescues MotorAbnormalities Resulting from Dopamine Deficiency in SPR-KO Mice

These results thus far indicate that rebound firing in VL thalamus issufficient to cause multiple motor abnormalities. Next, it wasinvestigated whether this thalamic mechanism is altered during PD-likemotor abnormalities. For this purpose, the GPm-VL circuit was examinedin a mouse model of dopamine deficiency, specifically, the sepiapterinreductase knockout (SPR-KO) mice constructed in Example 4. SPR catalyzesthe synthesis of tetrahydrobiopterin (BH4), a cofactor for tyrosinehydroxylase, the rate-limiting enzyme in the synthesis of dopamine. TheSPR gene is linked to a locus for familial Parkinson's disease (PARK3)of unknown genetic identity. Mutations in SPR are associated withL-DOPA-responsive dystonia, which is characterized by PD-like motordysfunction, including akinesia, rigidity, and tremor. Similarly, SPR-KOmice show motor abnormalities. However, their short lifespan (2-3 weeks)and severe health problems have limited analysis of their behavior andunderlying circuitry.

To improve the survivability of SPR-KO mice, their daily diet wassupplemented with tetrahydrobiopterin (BH4), which extended theirlife-span to old age. Older-aged KO mice had fewer dopaminergic axonfibers and lower striatal dopamine levels compared with WT mice (notshown). After cessation of BH4 feeding, mice rapidly (within 24 h)developed severe motor impairment, including akinesia, gait disturbance,tremor, and rigidity (not shown). Except for rigidity, all these motorproblems were ameliorated by the administration of L-DOPA, a standardtreatment for PD (not shown). These results indicate the utility ofSPR-KO mice with BH4 dietary supplementation as a reliable andreversible model of dopamine deficiency for analysis of PD-relatedneural circuitry.

Experimental Example 8: SPR-KO Mice not Administered the BH4 Diet ShowEnhanced Rebound Firing <8-1> In Vivo Spike Recordings in SPR-KO and WTMice

In vivo spike recording was performed to measure rebound firing inSPR-KO mice. For multi-unit recording, SPR-KO and WT mice wereanesthetized with urethane (1.35 g/kg, i.p.) and placed in a stereotaxicapparatus (David Kopf Instruments). Body temperature was maintained at37° C. using a temperature-control device (Homothermic Blanket System;Harvard Apparatus, USA). After making a single incision in the scalp,the skull was exposed, a hole was made above the VL region, and aquartz-coated tetrode (5-2 MS); Thomas Recording, Germany) was implantedinto the VL thalamus (−0.825 mm AP, −1.0 ML, −3.3-3.5 DV). Signals wereamplified ˜95-fold using an AC amplifier. Acquired signals were filteredwith a 300-5 kHz band-pass filter for measurement of multi-unit activityor a 0.50-50 Hz band-pass filter for the measurement of LFP, anddigitized at a sampling rate of 10 kHz (DT3010; Neuralynx, USA). Thelocation of the tetrode in the brain was confirmed by briefly dippingthe tip of the tetrode in fluorescent dye solution (DiI, 50 mg/ml;Sigma) before implantation. The position of the electrode in brainslices was visualized by fluorescence microscopy (IX51; Olympus) using arhodamine filter.

The rebound firing was identified from total multi-unit activitiesextracted from the spikes in each tetrode using threshold-based SpikeExtractor software (Neuralynx). The onset time for rebound firing wasidentified as the time when the firing rate was increased over the meanfiring rate, after the inhibitory period (over 50-ms) which shows lowerfiring rate than the mean. Because the reliable mean firing rate couldnot be computed within short duration, alternative method was utilizedin the recording dataset shorter than 30 s. The 50-ms window was shiftedevery 10 ms and the firing rate was computed in each window. In caseswhere the firing rate steadily increased in three consecutive windows,the starting point of the second window was defined as the point atwhich firing rate increased. Since the firing rates in the first windowwere lower than basal activity, this method successfully identify thepost-inhibitory activities.

To measure spontaneous rebound activity, spontaneous inhibition as adecrease in neuronal firing rate to a level 50% lower than the baselinefrequency was detected. After detecting such epochs of inhibition,multi-unit spikes in time for the 50 ms preceding (pink shading in FIG.23) and following the inhibitory event (FIGS. 23 and 24) were aligned.Consistent with the response of VL neurons to photostimulation of GPminputs (FIGS. 6(A) and 8(A)), inhibitory events in WT mice were followedby rebound firing (red arrowheads in FIG. 23). Rebound firing wasdramatically greater in SPR-KO mice (red arrowheads in FIG. 23(B)),particularly within the first 200 ms after inhibition (FIG. 24). Theintrinsic properties of VL neurons in SPR-KO mice were not significantlydifferent from those of WT neurons, as measured by their ability toinduce tonic and low-threshold burst spikes in brain slices (not shown).Instead, increased rebound firing appears to depend on greaterinhibitory drive to the VL. Thus, a dopamine-deficiency inducedinhibition appears to evoke rebound firing in a PD-like mouse model.

<8-2> In Vivo Spike Recordings in SPR-KO Mice Expressing Halorhodopsin

To address the role of GPm-VL inputs in the spontaneous rebound firingobserved in SPR-KO mice, as described in Example 5, GPm was infectedwith an adeno-associated virus (AAV) harboring eNpHR3.0 under thecontrol of the EF1α promoter. Illumination with green light (532 nm) wasused to photoinhibit GPm inputs, and multi-unit recordings were used todetermine the effects of photoinhibition (FIG. 25).

For the optogenetic inhibition of GPm-VL synapses in SPR-KO mice, therecording experiment was performed after injecting the virus to expresseNpHR3.0. Such photoinhibition of GPm inputs to VL substantially reducedrebound firing in KO mice (FIG. 26(A)).

To examine the effect of pharmacological inhibition of T-type calciumchannel, ethosuximide (150 mg/kg, i.p.) was treated during the recordingexperiment in SPR-KO mice. Treatment with ethosuximide (ETX; i.p. 150mg/kg), a blocker of T-type Ca²⁺ channels, also reduced spontaneousrebound firing of VL neurons in SPR-KO mice (FIGS. 26(B) and (C)),suggesting that in a dopamine-deficient state GPm inhibitory inputmediates rebound firing in the VL via T-type calcium channels.

Experimental Example 9: GPm-VL Inhibitory Inputs Mediate Motor Defectsin SPR-KO Mice

To assess the impact of GPm inputs on the motor deficits of SPR-KO mice,first, a loss-of-function optogenetics experiment was performed byphotoinhibiting the GPm-VL pathway.

Locomotor activity test, akinesia, rigidity test and tremor test wereperformed according to the methods described in Experimental Examples 1,<2-2> and <6-1>.

Photoinhibition of GPm inputs to the VL rescued the locomotor defect ofSPR-KO mice, allowing free movement (FIG. 27(A)). Additionally,photoinhibition decreased the locomotion latency of SPR-KO mice to thelevel of WT mice (FIG. 27(B)) (2.18±1.16 s vs. 2.19±0.71 s for WT mice)and reduced the time spent being immobile (FIG. 27(C)) (25%±16% vs.29%±10% for WT). Photoinhibition of GPm-VL inputs also decreased thelatency to release a horizontal bar by >35%, indicating efficientreversal of muscular rigidity (FIG. 28). The most significant changeobserved was a >50% reduction in tremor activity (FIG. 29(A)).

To examine the effect of pharmacological inhibition of T-type calciumchannel, ethosuximide (150 mg/kg, i.p.) was treated during tremor tests.In addition, the same tests were performed on the mice treated with theshRNA constructed in Example 6.

The abnormal motor functions of SPR-KO mice were ameliorated by reducingrebound firing, either via administration of ETX (FIG. 29(B)) orVL-specific knockdown of the Ca_(v)3.1 gene with targeted shRNA (FIG.29(C)). Taken together, these results indicate that GPm inputs mediate apowerful rebound firing of VL neurons, which underlies multiple forms ofPD-like motor dysfunction in SPR-KO mice.

Experimental Example 10: Rebound Excitability of VL Neurons in NormalConditions

Having established that rebound firing plays an important role in motordysfunction of dopamine-deficient SPR-KO mice, and that inhibition ofrebound firing rescues the PD-like motor impairments of these mice, VLneuron activity in WT mice was recorded to determine whether reboundfiring also occurs in physiological situations (FIG. 30).

<10-1> In Vivo Spike Recording During Natural Resting-RunningTransitions

Mice were anesthetized with avertin and placed in a stereotaxic device.Mice were prepared for recording on the running wheel by cementing acustom-designed head-plate with a window opening above the right VLthalamus to the skull with Super-Bond and dental cement. A small screw,serving as a ground electrode, was implanted in the skull above thecerebellum.

Mice were trained to voluntarily run on the wheel with their head fixed.The implanted head-plate was clamped to a holder device, which wasmodified from Royer et al. (2012). Training sessions were started morethan 2 days after surgery to allow recovery. Mice were mildlywater-deprived for 5-8 h before training. When a mouse ran more than theconditioned distance (about 15 cm, 6 blocks in the distance meter)within 5 s, it was rewarded with sucrose-in-water. Training wascontinued over 4 d (20 min/d) until each mouse had run more than 10times during the training session.

For recording awake, behaving status (FIG. 6), mice were anesthetizedwith isoflurane (1.5% in oxygen), and their heads were fixed to a holderdevice. The skull above the right VL thalamus (1.0 mmAP, −1.0 ML) wasdrilled to make a hole, and the dura was removed to allow insertion of asilicon probe (16 channels separated by 25 μm; NeuroNexus, USA). Theprobe was fixed to a micromanipulator and lowered into the brain. Afterreaching the VL thalamus, the hole was sealed with 1.5% liquid agarapplied at near body temperature. After the mice had completelyrecovered from the anesthesia, a recording session was started andtypically lasted for 60 min.

Mouse behavior and reward delivery were recorded as text in a computerfile, and video was recorded for detailed behavioral analysis using theCheetah acquisition system (Neuralynx). Neural signals were recordedwith the Cheetah 16-channel acquisition system (Neuralynx) and band-passfiltered at 0.5-50 Hz for LFP and 3-5 kHz for single-unit activity.

A 20 min period when mice were active was selected for analysis.Behaviors were classified into five categories; Rest (Nogo), Gotransition (Nogo to Go), Movement (Go), Stop transition (Go to Nogo),and other behaviors.

Rest and Movement were determined when the mice showed no movement(Rest) or actively ran (Movement). Other behaviors except runs, such aslicking and grooming, were not included in the Movement category.Complete moving and resting states were classified by removing periods 3s before and after active movement, and 1 s before and after subtlemovement.

Go and Stop transitions corresponded to initiation (Go transition) andtermination (Stop transition) of a run, respectively. Behavior wasconsidered a Go transition only when mice showed no movement for 3 sbefore movement initiation, and actively ran for more than 3 s aftermovement initiation. Behavior was considered a Stop transition only whenmice actively ran and slowed down for 3 s before movement termination,and did not move for 3 s after movement termination. The precise timesof Go and Stop transitions were determined by analyzing video recordings(29.97 frames/s) with frame-scale, and synced with a single-unitrecording system (TS clock in the Cheetah acquisition system).

Initially, the movement-related activity of VL neurons in WT mice wascompared during the natural transition between resting and running (FIG.31). Selectivity index values were determined based on the firing ratesassociated with specific movement states (No-go vs. Go) and subsequentlysorted in descending order. Nearly all thalamic neurons (94%) exhibitedstate-dependent changes in their activity. The majority of neuronsdisplayed a higher firing rate during the Go than No-go state. Thisrelationship was most apparent during transitions between the two states(FIG. 31) and is consistent with a rate-coding model, namely that adecrease in the average firing rate of VL neurons is associated withreduced locomotion.

Next, rebound firing during different behavioral states was examined byaligning the recordings of multi-unit spikes for 50 ms preceding andfollowing each inhibitory event, which again was defined as a 50% orgreater reduction in baseline firing frequency. Comparison of VLneuronal activity during four behavioral states (FIG. 32) revealed thestrongest rebound firing during the No-go state (asterisk in FIG. 32),occurring approximately 1-5 times per second. The normal incidence ofrebound firing was significantly lower than in the dopamine-deficientstate (˜40%, FIG. 24), indicating the involvement of a smaller number ofneurons involved in rebound firing in these mice compared to WT mice.Given that rebound firing of thalamic neurons activates muscles (FIGS. 6to 10 and FIGS. 16 to 22), the spontaneous rebound firing observed in WTmice may also stimulate the muscle activity required for maintainingtheir posture on a running wheel during the resting state. The PD-likesymptoms observed in SPR-KO mice may arise from increased rebound firingof neurons and subsequent dysfunctional muscle movement (FIGS. 23 to29). This hypothesis is consistent with the observation that PD symptomsare more severe during the resting state.

Decreased firing of VL neurons potentially involves a reduction in motoractivity and additional induction of post-inhibitory excitationstabilizes this ‘standstill’ by inducing sufficient muscle tension.However, excessive rebound firing may trigger pathological conditionsthat interfere with voluntary motor control, such as akinesia, rigidity,and tremor.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended Claims.

1. A method for treating Parkinson's disease, comprising inhibitingrebound firing of ventrolateral thalamus (VL) neurons, wherein theventrolateral thalamus (VL) neurons of a subject having Parkinson'sdisease are exposed to a T-type Ca2+ channel blocker in order to inhibitrebound firing of VL neurons.
 2. The method for treating Parkinson'sdisease according to claim 1, wherein the T-type Ca2+ channel blocker isa CaV3.1 gene expression inhibitor.
 3. The method for treatingParkinson's disease according to claim 2, wherein the CaV3.1 genecomprises the nucleotide sequence represented by SEQ ID NO:
 3. 4. Themethod for treating Parkinson's disease according to claim 2, whereinthe CaV3.1 gene expression inhibitor comprises siRNA, shRNA, or miRNAthat can bind to the CaV3.1 gene mRNA complementarily.
 5. The method fortreating Parkinson's disease according to claim 4, wherein the shRNAcomprises the nucleotide sequence represented by SEQ ID NO:
 4. 6. Themethod for treating Parkinson's disease according to claim 4, whereinthe siRNA, shRNA or miRNA is inserted in a vector.
 7. The method fortreating Parkinson's disease according to claim 6, wherein the vector isa DNA vector, a plasmid DNA vector, or a recombinant viral vector. 8.The method for treating Parkinson's disease according to claim 7,wherein the recombinant viral vector is a retrovirus, adenovirus,adeno-associated virus, or lentivirus vector.